| Popis: |
Background: Aging is a natural phenomenon in all organisms. The pace of aging and related manifestations depends on balance of formation and dismutation of reactive oxygen species (ROS) in the cell. Superoxide dismutase (SOD; EC 1.15.1.1) is an antioxidative enzyme that prevents the aerobic organisms from oxidative damage caused by free radicals that are generated during oxygen metabolism. This enzyme system is the key biomolecule of the cells having antiaging properties and increases the life span of organisms. Objective: The aim of present research was to isolate thermostable SOD producing bacterial strain and to increase its activity by optimizing the production and assay parameters for thermophilic bacterium using response Surface Methodology (RSM) and its identification and phylogenetic analysis using molecular techniques. Materials and methods: The water and soil samples were collected from thermal springs of different regions of Western Himalayas of India. Out of total 40 isolates, Bacillus sp. SPB-13 was screened for production of SOD on the basis of highest activity and identified using 16s rRNA gene sequencing. One Variable at a Time (OVAT) approach followed by central composite design was employed to analyze the effect of different media and reaction components, i.e., Glucose, casein, yeast extract, NaCl, buffer system, reaction temperature and enzyme concentration on SOD production. Results: One hyperproducer of SOD was isolated and found to be Bacillus licheniformis SPB- 13 (NCBI Accn. No. KX185731). Enzyme activity was enhanced from 80 U/mL to 120 U/mL after production and reaction process optimization. A significant increase in SOD activity of 40 U/mL was obtained after classical and statistical optimization. SOD remained fully active at 70°C and after metallotyping, found to be a member of Fe/Mn SOD family. Conclusion: Bacillus licheniformis SPB-13 produces a thermostable SOD with very good antioxidant activity. The production and reaction parameters have been optimized completely using classical and analytical methods to cut the production cost and to increase the yield of SOD. The antiaging potential of this enzyme can be exploited in cosmetics and pharma industry for antioxidative formulations and topical ointments. |
